The specific objectives of this project are: (1) understanding the mechanisms for the synthesis of ACh and related choline esters, (2) synthesizing the suitable inhibitors of choline acetyltransferase (ChA), which synthesizes ACh from choline and acetyl-coenzyme A (ACoA), (3) analyzing tissue extracts for ACh and related compounds by modern methods and (4) delineating cholinergic receptor sites using configurationally related agonists and antagonists. Our investigations have indicated the following: (a) L-Lactoyl-CoA could be formed from CoA and L-lactate (1 to 10 micromolar) in the presence of acetylthiokinase, ATP and Mg ions. Choline acetyltransferase could utilize L-lactoyl-CoA and choline for the formation of L-lactoylcholine. The concentrations of L-lactate in certain tissues will reach those concentrations (e.g., muscle, 0.44 to 40 m mol/kg; brain, 1 to 8 m mol/kg during exercise or in certain pathological states) at which the synthesis of L-lactoylcholine may proceed at an optimal rate. (b) The kinetic mechanism for the synthesis of ACh by the human placental and rat brain ChA's is possibly of the Theorell-Chance type. The availability of choline, the removal of CoA from the enzyme environment and the degree of saturation of the vesicles with ACh may regulate ACh synthesis in vivo. It is not possible to demonstrate the effect of a ChA inhibitor like bromoacetylcholine on ACh levels when vesicles were supersaturated with ACh. (c) Potential ChA inhibitors, which may be resistant to cholinesterases were prepared and these include 2(2- chloroethoxy)ethyl- trimethyl ammonium, 5-chloromethylfurfuryltrimethyl ammonium, N(2-chloropropionyl)3,3-dimethylbutylamine. Work on these compounds as ChA inhibitors in vitro and in vivo are in progress. (d) Gas chromatographic analysis indicated the presence of ACh (112 n mol/g wet tissue) and an unidentified compound (16 n mol/g and ACh equivalents) in placental extracts. Our investigations will lead to the understanding of the normal function of the cholinergic system (cholinergic receptor, ChA, occurrence of other choline esters) and its disfunction in certain pathological states.